The present invention relates to an enteric coated microgranule for optimally stabilizing lactic acid bacteria. In the present specification, the term xe2x80x9clactic acid bacteriaxe2x80x9d means the bacteria beneficial to health, which are present in human intestine and help to keep the peristalsis of intestine active.
The ingested lactic acid bacteria prevent the abnormal fermentation of food and activate the function of intestine, thereby improving the functional abnormality of intestine such as constipation, diarrhea, etc. and maintaining good care of health. Also, the addition of lactic acid bacteria to feedstuff can prevent the accumulation of gas, constipation, diarrhea, etc. caused by the abnormal fermentation inside the intestine of livestock which may result from the repetitive supply of the same feedstuff, which ultimately improves the quality of flesh and highly contributes to the development of dairy farming.
However, in spite of high usefulness and values of lactic acid bacteria, the actual use of lactic acid bacteria has many restrictions due to their acid-unstability. That is, since lactic acid bacteria are very unstable under pH 4, almost all the ingested lactic acid bacteria are destructed at the acidity of gastric juice (about pH 2). Therefore, only a trace amount of the ingested lactic acid bacteria (about one per million) can reach the intestine alive. As a result, much time and expenses are required to make lactic acid bacteria efficiently exhibit their functions in human intestine.
In order to overcome such a problem, a way to increase the amount of lactic acid bacteria which reach the intestine by using more than 10 times excess of bacteria has been proposed in the field of food and pharmaceutical industry. However, it is not a fundamental solution, but merely a very fragmentary and wasting, temporary remedy. Further, food containing microcapsules wherein lactic acid bacteria are mixed with fat, emulsifying agent and protective material and then encapsulated has recently been reported, the purpose of which is to increase the ratio of lactic acid bacteria arrived at the intestine by making the bacteria survive in gastric juice (see, Korean Patent Laid-open Publication No. 97-25405). However, according to the experimental result, it has been identified that upon ingestion of such encapsulated lactic acid bacteria their coating is disintegrated within 30 minutes regardless of the circumstance being gastric juice or intestinal juice. It can also be noted from other experiments that the commercially available lactic acid bacteria coated with gelatin as the coating base are not disintegrated for more than 10 hours in any circumstances without difference on the specificity to gastric juice or intestinal juice (see, Experimental Example 1). It appears that this is because the material used as the coating base for lactic acid bacteria is a conventional one which does not react sensitively to the property of the gastric or intestinal juice. Further, numerous organic solvent-based coating methods utilizing various polymers have been reported in the general pharmaceutical field (see, PCT/JP94/001675, Japanese Patent Appln. Nos. 91-235667, 92-364123, 92-41434, 93-186335, 93-186336, etc.). However, such coating techniques are not satisfactory to protect lactic acid bacteria from gastric juice. Particularly, if organic solvent is used as a solvent of the coated preparation or if the coating process is carried out at a high temperature of more than 55xc2x0 C., the actual survival rate of lactic acid bacteria in human body is much less than the expected value.
On the other hand, it has been tried to develop variant strains of lactic acid bacteria, which have a high acid-resistance. However, this approach requires greater time and cost, and has worse effect than the coating methods.
The present inventors have intensively studied about enteric coating technique which gives some usable merits in view of the stability of lactic acid bacteria and in the economical view. As a result, we have found that when lactic acid bacteria are first coated with a specific water-miscible coating material and then, if desired, second coated with a conventional controlled-release coating material, the destruction of lactic acid bacteria during the procedure for preparing the coated granule can be greatly reduced and, furthermore, the coated granule capable of delivering lactic acid bacteria contained therein to the target organ in which lactic acid bacteria actually display their function, i.e. intestine, by safely protecting lactic acid bacteria from the attack of gastric juice can be produced. Thus, we have completed the present invention. In the present invention, since the coated granule contains active lactic acid bacteria in a high ratio and is very sensitive to acidity, the bacteria contained therein can survive under human gastric circumstance and the granule then can be disintegrated rapidly in the intestine.
Therefore, it is an object of the present invention to provide an enteric coated microgranule specially designed so as to display the function of lactic acid bacteria in the intestine by optimally stabilizing lactic acid bacteria contained in the granule.
The coated granule containing lactic acid bacteria according to the present invention is more specifically explained in below.
The coated granule containing lactic acid bacteria according to the present invention can be prepared by first coating the lactic acid bacteria-containing seed with a water-miscible coating material at low temperature and, if desired, then subjecting the first coated product to the second coating with a controlled-release coating material. In the present invention, the destruction of lactic acid bacteria during the procedure for preparation can be minimized by conducting the first coating with a water-miscible material at low temperature.
In the present invention, one or more strains beneficial to human being, which are selected from the group consisting of Streptococcus genus, Lactococcus genus, Leuconostoc genus, Pediococcus genus, Enterococcus genus, Lactobacillus genus and Bifidobacterium genus can be used as the lactic acid bacteria strain.
The water-miscible coating material which can be used for the first coating includes sodium alginate as the main ingredient of seaweed (e.g., brown seaweed) extract, alginic acid, polymethylmethacrylate [Eudragit L30D, Eudragit LS30D, Kollicoat MAE 3DP (manufactured by BASF Co.), etc.], wheat protein, soybean protein, methylcellulose (MC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose [HPMC; pharma coat, aqua coat, etc.], polyvinylacetatephthalate [Sureteric; manufactured by Colorcon Co.], gums, for example, guar gum, locust bean gum, xanthan gum, gellan gum, arabic gum, etc. Since these water-miscible coating materials are water-soluble or water-dispersible, it is advantageous that the first coating procedure can be conveniently carried out by using water as a solvent. This is very important in view of the fact that any organic solvent which is not only harmful to human body but also fatal to the stability of lactic acid bacteria is not used for the coating procedure, and therefore, the problem of removing the residual organic solvent is completely solved. This is a characteristic advantage of the present invention as distinct from the prior techniques which necessarily require the use of organic solvents to dissolve high-molecular substances as a coating material. In the present invention, sodium alginate is preferably used as the water-miscible first coating material. The reason is that since sodium alginate is water-soluble and its aqueous solution is neutral, it is much more advantageous for the stability of lactic acid bacteria.
The seed used for the coating procedure can be either lactic acid bacteria themselves or a mixture of lactic acid bacteria and one or more additive substances selected from the group consisting of starch, lactose, oligosaccharides, glycoalcohols, calcium gluconate, calcium lactate and gluconic acid. These additives are added for the purpose of diluting lactic acid bacteria in a desired ratio, activating only lactic acid bacteria while suppressing other bacteria strains, or improving the proliferation of lactic acid bacteria.
In the first coating procedure, the water-miscible coating material is preferably used in an amount of 1 to 80% by weight with respect to the seed.
Although the first coated granule of lactic acid bacteria as prepared above is sufficiently effective by itself, but it can be more effectively used after second coating with a conventional controlled-release coating material. Therefore, an enteric coated microgranule with both the first and second coatings is also included within the scope of the present invention.
As the second coating material, the controlled-release coating material, particularly an enteric coating material commonly used in pharmaceutical field; or a coating material for swelling such as carbopol or arabic gum; and other controlled-release coating materials can be used. More specifically, corn protein extract (described in USP/NF) and artificial processed materials thereof, such as for example, Zein-DP or prolamin, sodium alginate, alginic acid, polymethylmethacrylate, for example, Eudragit L30D, Eudragit LS30D, Kollicoat MAE 3DP (manufactured by BASF Co.), etc., shellac, hydroxypropylmethylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose (HPMC), hydroxy-propylmethylcelluloseacetatesuccinate (HPMCAS), carboxymethylcellulose (CMC), hydroxypropylcellulose (HPC), celluloseacetatephthalate (CAP), polyvinylacetatephthalate [Sureteric(Colorcon Co.)], ethylcellulose (EC), methylcellulose (MC), soybean protein or wheat protein (they are registered as Food Additives), chitin, chitinic acid, agar, carrageenan, pectin, carbopol, or gums, such as for example, guar gum, locust bean gum, xanthan gum, gellan gum, arabic gum, etc. can be mentioned. Among them, one or more selected from the group consisting of corn protein extract, hydroxypropylmethylcellulose phthalate (HPMCP) and shellac are preferably used as the second coating material.
In conducting the second coating procedure, one or more materials selected from the coating materials as mentioned above are used in an amount of 1 to 95% by weight with respect to the first coated granule. Particularly, when the enteric coating material commonly used in the pharmaceutical field is used, it is used in an amount ranging from 1 to 40% by weight; or when other coating materials for swelling are used, it is used in the amount ranging from 30 to 95% by weight. The kind and amount of the coating material may be appropriately determined by a person skilled in the relevant technical field, considering the property of coating material and the purpose of using the coating material.
Contrary to the first coating wherein only water is used as a solvent for the protection of lactic acid bacteria, the second coating may use one or more various solvents selected from water, alcohol; acetone, acetonitrile, methylene chloride, ether, hexane, chloroform, 1,4-dioxane, tetrahydrofuran, dimethylsulfoxide, ethyl acetate and methyl acetate. In case the coating material is hardly dissolved in the solvent, if required, a pH regulator such as acetic acid, hydrochloric acid, phosphoric acid, various buffer solutions, citric acid, tartaric acid, malic acid, etc. can be used to adjust the pH to the desired range thereby improving the solubility of the coating material. This can be easily carried out by a person skilled in the relevant art.
When both of the first and second coatings are conducted, the coating materials used in the respective steps should be different from each other. If desired, one or more plasticizers selected from a group consisting of polyethyleneglycols, myvacet, propyleneglycol, glycerine, triethyl citrate, triacetin, cetyl alcohol and stearyl alcohol can be used in the first or second coating procedure. In this case, the plasticizer is preferably used in an amount of 1 to 50% by weight with respect to the coating material as used.
To optimally stabilize the lactic acid bacteria in preparing the coated granule, the present invenors have utilized a process in which (a) the seed containing lactic acid bacteria is suspended and, at the same time, spray-coated with a coating-solution, or (b) the seed suspended in the coating-solution is dispersed into a chamber. Thus, the present invention can be carried out more preferably by applying such processes.
The coating process may be carried out by using a fluidized bed granulator, CF-granulator and the like, preferably a fluidized bed granulator (SFC-MINI, Freund co., Japan). When such a granulator is used, the temperature of the introduced air is preferably maintained in the range of 40 to 70xc2x0 C. The temperature of granule in the granulator at each step should be kept more than 20xc2x0 C. to prevent the granule from absorbing moisture from the ambient atmosphere and coagulating with each other. Preferably, the temperature of granule is maintained from 25 to 55xc2x0 C. throughout the whole procedures since lactic acid bacteria may be destroyed at the temperature exceeding 55xc2x0 C.
The present invention will be more specifically explained by the following examples and experimental examples. However, it should be understood that the examples are intended to illustrate but not to in any manner limit the scope of the present invention.